Synthesis of a highly thermostable insulin by phenylalanine conjugation at B29 Lysine

Globally, millions of diabetic patients require daily life-saving insulin injections. Insulin heat-lability and fibrillation pose significant challenges, especially in parts of the world without ready access to uninterrupted refrigeration. Here, we have synthesized four human insulin analogs by conjugating ε-amine of B29 lysine of insulin with acetic acid, phenylacetic acid, alanine, and phenylalanine residues. Of these, phenylalanine-conjugated insulin, termed FHI, was the most stable under high temperature (65 °C), elevated salt stress (25 mM NaCl), and varying pH levels (ranging from highly acidic pH 1.6 to physiological pH 7.4). It resists fibrillation for a significantly longer duration with sustained biological activity in in vitro, ex vivo, and in vivo and displays prolonged stability over its native counterpart. We further unravel the critical interactions, such as additional aromatic π-π interactions and hydrogen bonding in FHI, that are notably absent in native insulin. These interactions confer enhanced structural stability of FHI and offer a promising solution to the challenges associated with insulin heat sensitivity.

(Boc)2O.The aqueous layer was then acidified with 1N HCl to pH 4-5.After acidification aqueous layer was extracted with ethyl acetate (2 times) and combined organic layer was washed with brine solution followed by drying of organic layer over anhydrous sodium sulphate.Finally, the solvent was evaporated under reduced pressure to get Boc-Phe-OH as white solid which is used directly in the next step without purification.
Boc-L-Phenylalanine (1.6 gm, 6.05 mmol) and N-hydroxysuccinimide (1.2 equiv., 0.83 gm, 7.26 mmol) were dissolved in dry dichloromethane and cooled to 0 ºC.To this small amount of tetrahydrofuran was added to achieve complete solubility.To this 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or EDC.HCl (1.5 equiv., 1.73 gm, 9.075 mmol) was added directly to the reaction under cold condition.The reaction was stirred for 1 h at 0 °C and then stirred overnight at RT under anhydrous condition.Reaction was monitored using TLC and upon completion solvents including dichloromethane as well as tetrahydrofuran were evaporated.The residual was then dissolved in ethyl acetate and afterwards washed with water.Collected organic layer was treated with brine followed by drying of organic layer over anhydrous sodium sulphate and evaporated under reduced pressure to give Boc-Phe-OSu as white solid.yield (1.7
The reaction was stirred for 1 h at 0 °C and then stirred overnight at RT under anhydrous condition.
Reaction was monitored using TLC and upon completion extra dichloromethane was added and afterwards washed with water.Collected organic layer was washed with saturated bicarbonate solution.
Collected organic layer was further treated with brine solution followed by drying of organic layer over anhydrous sodium sulphate and evaporated under reduced pressure to give succinimidy ester of acetic acid (Ac-OSu) as white solid.Yield (0.246 gm, 1.57 mmol, 80.1 %).

High performance liquid chromatography (HPLC) analysis
HPLC analyses were performed with a HPLC system (Agilent technologies 1260 infinity) equipped with a quaternary pump (G1311B), auto liquid sampler (G1329B), Diode array detector (G1315D) and analytical scale-fraction collector (G1364C).Instrument control, data acquisition and data analysis was performed using a ChemStation software (Agilent Technologies, Workingham, UK).A ZORBAX Eclipse plus C18 (250 x 4.6 mm) column with 5 µm particle size at room temperature was used.Mobile phase consisted of acetonitrile/water with 0.1 % TFA and the flow rate was 1.0 mL/min.Injection volume was

Insulin activity long cold storage
Commercially available wild type insulin is known to lose its activity when stored in refrigeration for long durations, where it tends to aggregate and form toxic fibrils. 1 To test the efficacy of the modified insulin analog FHI as compared to that of HI, we stored both HI and FHI at 4 ºC for 2 months.Serum starved HEK293T cells were then treated with both HI and FHI at a concentration of 0.5 µmol for 30 minutes and the level of phosphorylated AKT was measured using western blot analysis for specific antibody as a read out of insulin activity.Interestingly, the modified insulin FHI retained its activity in the present treatment conditions as compared to the untreated controls whereas the human recombinant insulin HI failed to show any significant difference with the untreated control in Akt phosphorylation suggesting that FHI is far more stable than the wild type insulin.Experiments were carried out at 25 ± 0.1 °C using quartz cuvette with a path length of 1 mm.

HPLC analyses of the samples after heat treatment
To quantitatively evaluate the soluble populations of both HI and FHI after heat treatment, we performed HPLC analysis in Agilent 1260 infinity machine using Poroshell-120 EC-C18 (150 × 3 mm, 2.7 μm) reverse phase column.We plotted the area under the curve (AUC) of their corresponding peaks in the HPLC chromatograms for both fresh and heated (65 °C) samples at acidic (pH 1.6, Fig. S32) and physiological (pH 7.4, Fig. S33) pH levels.
10 μL and the column effluent was monitored at 220 nm.Program set with the initial mobile phase composition of acetonitrile/water (10:90) with gradual increase in acetonitrile concentration from 10 % to 70 % within 20 min of run.Further acetonitrile concentration was increased from 70 % to 95 % within next 5 min of run.

Figure S1 :
Figure S1: Analytical HPLC chromatograms of reaction mixtures (reaction between HI and Boc-Phe-OSu) after 1 h at different pHs.

Figure S24 :
Figure S24: Comparative in-vitro efficacy of FHI in HEK293T cell line upon refrigeration at 4 °C for 2 months.a. Representative immunoblots showing the relative levels of P-AKT (Ser 473) in the

Figure S26 .
Figure S26.Aromatic interactions in FHI between B29Lys(Phe) and other aromatic residues.a. Distance between COM of phenyl rings of B29Lys(Phe) and A19Tyr, B24Phe, B25Phe, and B26Tyr with time, and b. their probability distribution.

Figure S27 .
Figure S27.Hydrogen bonding interactions between B29 and A3 residues.a. Distance between backbone carbonyl oxygen of B29Lys(Phe)/B29Lys and backbone amide hydrogen of A3Val in FHI and HI residues with time, and b. their probability distribution.

Figure S28 .
Figure S28.Total no. of a. H-bonds and b. π-π aromatic interactions in HI and FHI protein with time.Solid-thick lines indicate the block average of every 10 ns data, while the thin lines indicate the instantaneous values.

Figure S29. a .S17 9 .
Figure S29.a. Electrostatic energy (Eelec) and b. van der Waals (Evdw) energy in HI and FHI protein with time.Solid-thick lines indicate the block average for every 10 ns data, while the thin lines indicate the instantaneous values.

Figure S32 .
Figure S32.HPLC analysis and quantification of fresh as well as heated (65 °C) samples of HI and FHI at pH 1.6.

Figure S33 .S20 12 .
Figure S33.HPLC analysis and quantification of fresh as well as heated (65 °C) samples of HI and FHI at pH 7.4.

Table S1 .
B29 Lys modifications in insulin

Comparison of current work with previous studyTable S2 .
Comparison of the current study with the insulin analog reported in Protein J., 2023, 42,